This proposal focuses on how spores are formed by Bacillus subtilis. B. subtilis has become a paradigm for the study of spore formation, and of cell differentiation in prokaryotes, because of the ease of its genetic manipulation. All the key regulators of B. subtilis spore formation are also found in other spore forming species including the pathogens B. anthracis, Clostridium tetani, C. perfringens, C. botulinum and C. difficile, the causes of anthrax, tetanus, gas gangrene, botulism and colitis following antibiotic therapy, respectively. The highly resistant spores are critical to the survival of theses species in nature. Conclusions from a study of B. subtilis will have general validity for these species. Spore formation involves a characteristic division into two different cell types, the mother cell and the prespore. The prespore is engulfed by the mother cell and develops into the mature spore. Spore formation requires the action of four RNA polymerase factors, ?F and then ?G in the prespore and ?E and then ?K in the mother cell. It presents a fundamental problem of developmental biology: how is gene expression coordinated with morphological change? The proposal addresses this problem. When the ?F-directed signal from the prespore to activate ?E in the mother cell is delayed, a novel developmental path is taken, in which two spores are formed within one mother cell. This behavior opens up the proposed study of how chromosome replication and growth are controlled during sporulation. It facilitates a study of how genetically identical organisms in the same population can follow different developmental paths. Translocation of the chromosome into the prespore follows activation of ?F. It is proposed to study how completion of translocation leads to engulfment of the prespore by the mother cell and activation of ?G in the engulfed prespore. PUBLIC HEALTH RELEVANCE: This proposal focuses on the fundamental problem of how gene expression is coordinated with morphological changes during formation of spores by Bacillus subtilis. All the key sporulation regulatory genes are also found in related pathogens, including B. anthracis, Clostridium tetani, C. perfringens, C. botulinum and C. difficile. Emphasis is placed on how altering the timing of signaling can change the developmental path taken.